Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry

Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.     

Characterization of archaeological beeswax by electron ionization and electrospray ionization mass spectrometry

To better detect and identify beeswax in ancient organic residues from archaeological remains, we developed a new analytical methodology consisting of the analysis of (i) the trimethylsilylated organic extract by GC/MS and (ii) the crude extract by ESI-MS. Selective scanning modes, such as SIM or MRM, permit separate quantification of each chemical family (fatty acids, monoesters, monohydroxyesters, and diesters) and allow an improvement in sensitivity and selectivity, allowing the crude extract to be treated without further purification. GC/MS (SIM) was revealed to be a powerful method for the detection of components, with a detection limit down to a total lipid extract in the range of approximately 50 ng in a complex matix, such as archaeological degraded material, whereas ESI-MS/MS is instead used for the detection of nonvolatile biomarkers. Identification by GC/MS (SIM) and ESI-MS/ MS (MRM) of more than 50 biomarkers of beeswax in an Etruscan cup at the parts-per-million level provides the first evidence for the use of this material by the Etruscans as fuel or as a waterproof coating for ceramics.     

Determination of dissolved metal species by electrospray ionization mass spectrometry

The distribution of metal species in solution was determined using flow injection electrospray ionization mass spectrometry. Complexes formed by selected metal ions with added organic ligands in 50:50 water/acetonitrile and 50:50 water/methanol under acidic, neutral, and basic conditions were detected using electrospray ionization conditions optimized to best represent solution-phase interactions. Metal species containing acetate, nitrate, and solvent molecules predominated in acidic solution but became less abundant at higher pH. Interactions between metal ions and added organic ligands became more selective with increasing pH, showing the expected preference of hard and soft ligands for metal ions of the corresponding type. Species distributions also tended toward larger complexes as pH increased. Overall ion yield was greater for aqueous acetonitrile than for aqueous methanol solutions; however, reduction of copper(II) in aqueous acetonitrile resulted in the detection of copper(I) complexes for certain ligands. Experimental results for copper(II) and 8-hydroxyquinoline in 50:50 water/methanol showed good agreement with aqueous speciation predicted using the thermodynamic equilibrium model MINEQL. Detection of neutral complexes was achieved by protonation, deprotonation, or electrochemical oxidation during electrospray.     

A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.     

Investigation of quadruplex oligonucleotide-drug interactions by electrospray ionization mass spectrometry

Selectivity, binding stoichiometry, and mode of binding of Tel01, distamycin A, and diethylthiocarbocyanine iodide (DTC) to the parallel stranded G4-quadruplex [d(T2G5T)]4 were investigated by ESI-MS. The first drug/quadruplex complexes observed by ESI-MS are described. Tel01, distamycin A, and DTC all form complexes with quadruplex DNA, but only Tel01 is completely selective for quadruplex versus duplex oligonucleotide under the conditions employed. Previous solution determinations of the binding mode of Tel01 and distamycin A to quadruplex oligonucleotides indicate that Tel01 interacts through end-stacking with guanine tetrads of quadruplex DNA, while distamycin A interacts by binding to quadruplex grooves. When these two different drug/quadruplex complexes are subjected to collisionally activated dissociation in a mass spectrometer, the observed fragmentation patterns are distinct. Tel01/quadruplex complexes undergo facile loss of drug and dissociation to single-strand oligonucleotide ions, while distamycin/quadruplex complexes fragment into single-strand oligonucleotide ions in which the drug molecule is retained. Dissociation patterns for DTC/quadruplex complexes are similar to those of distamycin; therefore, it is concluded that DTC interacts with [d(T2G5T)]4 through groove-binding. These ESI-MS results are applicable to both the identification and characterization of G-quadruplex interactive agents and may also be useful in probing unusual DNA structures.     

Desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Droplet dynamics and ionization mechanisms in desorption electrospray ionization mass spectrometry

A droplet pickup and other mechanisms have been suggested for the ionization of biomolecules like peptides and proteins by desorption electrospray ionization. To verify this hypothesis phase Doppler particle analysis was used to study the sizes and velocities of droplets involved in DESI. It was found that impacting droplets typically have velocities of 120 m/s and average diameters of 2-4 microm. Small differences in sprayer construction influence the operating conditions at which droplets of these dimensions are produced. Under these conditions, the kinetic energy per impacting water molecule is less than 0.6 meV and sputtering through momentum transfer during collisions or ionization by other electronic processes is unlikely. Droplets arrive at the surface with velocities well below the speed of sound in common materials, thereby excluding the possibility of ionization by shockwave formation. Some droplets appear to roll along the surface, increasing contact time and presumably the amount of material that is taken up into droplets during conditions typical of the DESI experiment.     

Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry

An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.     

New developments in biochemical mass spectrometry: electrospray ionization

The principles, development, and recent application of electrospray ionization-mass spectrometry (ESI-MS) to biological compounds are reviewed. ESI-MS methods now allow determination of accurate molecular weights for proteins extending to over 50,000, and in some cases well over 100,000. Similar capabilities are being developed for oligonucleotides. The instrumentation used for ESI-MS is briefly described and it is shown that, although ionization efficiency appears to be uniformly high, detector sensitivity may be directly correlated with molecular weight. The use of tandem mass spectrometry (e.g., MS/MS) for extending collision-induced dissociation (CID) methods to the structural studies of large molecules is described. For example, effective CID of various albumin species (molecular weight approximately 66,000) can be obtained, far larger than obtainable for singly charged molecular ions. The combination of capillary electrophoresis, in both free solution zone electrophoresis and isotachophoresis formats, as well as microcolumn liquid chromatography with ESI-MS, provides the capability for on-line separation and analysis of subpicomole quantities of proteins. These and other new developments related to ESI-MS are illustrated by a range of examples. Fundamental considerations suggest even more impressive developments may be anticipated related to detection sensitivity and methods for obtaining structural information.     

Metal complexation of thiacrown ether macrocycles by electrospray ionization mass spectrometry

In the present study, electrospray ionization mass spectrometry is used to evaluate the metal-binding selectivities of an array of novel caged macrocycles for mercury(II), lead(II), cadmium(II), and zinc(II) ions. In homogeneous methanol/chloroform solutions as well as extractions of metals from aqueous solution by macrocycles in chloroform, it is found that the type of heteroatom (S, O, N), cavity size, and presence of other substituents influence the metal selectivities. Several of the macrocycles in this study bind mercury ion very selectively and efficiently in the presence of many other metal ions and have an avidity toward mercury that was tunable by the size and combination of heteroatoms in the macrocycle ring and the number of cage groups attached. The extraction mechanism was further investigated by determining the variation in extraction selectivity as a function of the counterions of the mercury salts.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Frequency dependence of alternating current electrospray ionization mass spectrometry

The novel effects resulting from the entrainment of low mobility ions during alternating current (ac) electrospray ionization are examined through mass spectrometry and voltage/current measurements. Curious phenomena such as pH modulation at high frequencies (>150 kHz) of an applied ac electric field are revealed and explained using simple mechanistic arguments. Current measurements are utilized to supplement these observations, and a simplified one-dimensional transient diffusion model for charge transport is used to arrive at a scaling law that provides better insight into the ac electrospray ionization process. Moreover, because of the different pathway for ion formation in comparison to direct current (dc) electrospray, ac electrospray (at frequencies >250 kHz) is shown to reduce the effects of ionization suppression in a mixture of two molecules with different surface activities.     

Enhanced detection of flavonoids by metal complexation and electrospray ionization mass spectrometry

Metal complexation with the use of an auxiliary ligand is explored as an alternative to conventional protonation or deprotonation for analysis of a series of flavonoids by electrospray ionization mass spectrometry. Use of a neutral auxiliary ligand, 2,2'-bipyridine, results in formation of [MII(flavonoid - H)bpy]+, ternary complexes with intensities that are 2 orders of magnitude greater than the corresponding protonated flavonoids and up to 1.5 orders of magnitude greater than the deprotonated flavonoids, based on confirmation by collisionally activated dissociation patterns. The formation of ternary complexes with six divalent transition metals, Co2+, Ni2+, Cu2+, Zn2+, Mn2+, and Fe2+ were compared. Cu2+ resulted in the most intense complexes and simplest mass spectra, while Co2+ gave the second most intense spectra and also produced two key products that could be useful for a selected ion monitoring strategy. Complexation with iron(III) bromide is also investigated to explore the feasibility of using triply charged metals.     

Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.     

Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.     

Identifying specific small-molecule interactions using electrospray ionization mass spectrometry

A simple method for establishing whether complexes composed of small molecules detected by electrospray ionization mass spectrometry (ES-MS) originate from specific interactions in solution or nonspecific binding during the ES process is described. The technique, referred to as the nonspecific probe method, exploits the tendency of small molecules to bind nonspecifically to macromolecules during the ES process to establish the presence of specific noncovalent interactions. To implement the method, a macromolecule probe (P(NS)), which does not bind specifically to any of the components present in solution, is added prior to ES-MS analysis. The existence of specific small-molecule complexes is determined from an analysis of the measured distributions of the small molecules bound nonspecifically to P(NS). The principal assumption on which this methodology is based is that nonspecific binding of small molecules and their complexes to P(NS) during ES is a statistical (random) process. A mathematical framework for establishing the presence of specific heterocomplexes is presented. The reliability of the method for distinguishing specific from nonspecific small-molecule interactions is illustrated for peptide-antibiotic and metal ion-ligand interactions in water.     

Structural characterization and detection of kale flavonoids by electrospray ionization mass spectrometry

Sensitive and precise analytical methods are needed for flavonols, a subclass of flavonoids that has strong antioxidant activity. We report an improved method for identifying the predominant flavonols, quercetin and kaempferol, by collisionally activated dissociation (CAD) and quantifying them by high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) in the selected ion monitoring mode. Practical applications of the method were demonstrated using several kale and biological samples. Two commercial kale samples were found to have 77 or 244 ppm quercetin and 235 or 347 ppm kaempferol (ppm = microg of quercetin/g of kale or microg of kaempferol/g of kale by fresh weight, 5-15% relative standard deviation). Blanching was found to reduce the flavonols to approximately 60% of the levels found in the unblanched kale. Isotopically labeled kale (cultivar Vates) grown in a greenhouse under an atmosphere of (13)CO(2) was found to have much lower flavonol levels. UV-A and UV-B supplementation during kale growth in the greenhouse was found to enhance both quercetin and kaempferol levels in Vates kale. The UV-B-supplemented kale not only produced more flavonols but the quercetin-to-kaempferol ratio was also higher than the UV-A-supplemented or the nonsupplemented kale. Recovery of flavonols from kale was approximately 60% based on spike and recovery trials with rutin, a glycoside of quercetin. Recovery of flavonols from biological samples spiked with rutin ranged from 96% for urine to 70% for plasma. Compared to UV detection, ESI-MS in the deprotonation mode provided lower detection limits, and both higher sensitivity and selectivity, in addition to structural characterization of the kale flavonols by CAD.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.